nptII Expression Research Articles

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

The DFR locus: A smart landing pad for targeted transgene insertion in tomato.

Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2days.

Factors affecting Agrobacterium-mediated transformation efficiency of kumquat seedling internodal stem segments

Kumquat (Fortunella crassifolia Swingle.) has a short juvenile phase of 2–3 years and it can flower and bear fruit several times per year. It is, therefore, helpful for citrus to evaluate functional genes rapidly. In the present experimentation, a comprehensive and systematic study was performed to improve the transformation efficiency of kumquat, which was usually less than 4.1% in previous reports. Several factors, including explant status, Agrobacterium infection procedure, and co-cultivation conditions, were tested by using single factors or orthogonal experimental design. The higher regeneration and transformation rates were obtained from the seedlings cultured for 25days in darkness followed by 10days under light. The Agrobacterium cells suspended with co-cultivation medium to a final concentration 1×109 cfu/ml (OD600 value of 0.5) and immersing explants in Agrobacterium inoculum for 30min, resulted in better infection efficiency. The results indicated that the following conditions could improve the transformation efficiency. These treatments included adding 100μM AS into inoculum and co-cultivation media, placing internodal stem segments by end to end on co-cultivation medium for 3days at 25°C and placing co-cultivation medium in pH 5.4 with sterile filter paper support. With all these improvements, GUS-positive buds were recovered at the positive rate of 23.6% on selection medium. Polymerase chain reaction (PCR) analysis using gene-specific primers further confirmed the integration of the transgene for in vitro grafted plantlets at the transformation rate of 10.35%. Normal expression of nptⅡ and FLP genes was observed by RT-PCR and Real time q-PCR analysis.

Enhancing T-DNA Transfer Efficiency in Barley (Hordeum vulgare L.) Cells Using Extracellular Cellulose and Lectin.

A major limitation of transforming barley tissues by Agrobacterium tumefaciens is the low frequency of T-DNA transfer due to recalcitrance of barley as a host. The effect of extracellular cellulose and lectin on Agrobacterium transformation efficiency was investigated in this study. Barley callus cultures were transformed with the AGL1 strain containing the vector pBI121 in the presence of 10mgmL(-1) cellulose or 0.001, 0.05 and 0.1mgmL(-1) lectin. Addition of cellulose significantly (P ≤ 0.05) increased the number of GUS spots by 50% compared to standard conditions in the presence of only 200μM acetosyringone (AS). Frequency of G418-resistant aggregates on the surfaces of callus cultures was 29 and 71.5%, following AS and AS + cellulose treatments, respectively, after 4weeks of selection. Presence of 0.05 or 0.1mgmL(-1) lectin also increased the number of GUS spots and frequency of G418-resistant cells in the selection period, but the increase in blue spots was not significant. We examined the effect of lectin and cellulose on bacterial attachment to callus tissues. Both cellulose and lectin were found to have a significant positive effect on the numbers of bacteria attached to barley callus. Epifluorescence microscopy revealed that Agrobacterium cells had accumulated in the scaffolds of irregular fibrous cellulose with a mean particle size of 200μm. Expression of nptII in transformed callus lines confirmed the stable transformation of the gene. Our study showed for the first time the binding of Agrobacterium cells to fibrous cellulose and also demonstrated how polysaccharides and glycoproteins can be used to improve T-DNA transfer in monocotyledon transformation procedures.

Sugarcane (Saccharum spp. hybrids).

Genetic transformation of sugarcane has a tremendous potential to complement traditional breeding in crop improvement and will likely transform sugarcane into a bio-factory for value-added products. We describe here Agrobacterium tumefaciens-mediated transformation of sugarcane. Embryogenic callus induced from immature leaf whorls was used as target for transformation with the hypervirulent Agrobacterium strain AGL1 carrying a constitutive nptII expression cassette in vector pPZP200. Selection with 30 mg/L geneticin during the callus phase and 30 mg/L paromomycin during regeneration of shoots and roots effectively suppressed the development of non-transgenic plants. This protocol was successful with a commercially important sugarcane cultivar, CP-88-1762, at a transformation efficiency of two independent transgenic plants per g of callus.

Gene expression of beta carotene genes in transgenic biofortified cassava.

Cassava is an important food for millions of people around the world. However, cassava is deficient in protein, iron, zinc, pro-vitamin A and vitamin E. Cassava biofortified with pro-vitamin A can help reduce Vitamin A Deficiency among the undernourished communities that rely upon it for sustenance. BioCassava Plus project has developed transgenic cassava that expresses beta carotene in roots using root specific patatin promoter. This study aimed at confirming expression of nptII, crtB and DXS genes. Leaf and roots samples were obtained from confined field trial at KARI Alupe at 12 months after planting. RNA was isolated from cassava roots and leaves using a modified Dellaporta protocol, analyzed for expression of DXS, crtB and the selectable marker, nptII using the one step RT-PCR kit (Qiagen) and analyzed through gel electrophoresis. DXS, crtB and nptII genes were expressed in the roots as anticipated. On the contrary, DXS and crtB genes were also expressed in the leaves of the transgenic cassava despite the use of root specific patatin promoter. This finding indicates that the promoter confers expression in leaves too. Intensive molecular screening of the biofortified transgenic cassava is important for risk assessment that informs on integrity of the promoter gene and confirms expression of the beta carotene genes.

An Agrobacterium-mediated transformation system of pyrethrum (Tanacetum cinerariifolium) based on leaf explants

Pyrethrum (Tanacetum cinerariifolium) is well-known for the production of natural pyrethrin insecticides, which are isolated from the dried flower heads. To allow genetic analysis and improvement of pyrethrum in terms of yield and quality by molecular breeding a transformation protocol would be desirable. To achieve this, first an efficient direct shoot regeneration system was set up. Leaf explants formed shoots within 4 weeks on MS medium supplemented with 2.0mgl−1 6-BA and 0.2mgl−1 IBA. The best regeneration frequency obtained was 87% with an average number of 2 shoots per explant. Subsequently, a genetic transformation protocol was developed. Kanamycin added to the medium significantly reduced explant regeneration, and no green shoots were formed after 4 weeks incubation at 10mgl−1 kanamycin. Severe phytotoxic effects of cefotaxime were observed at concentrations >300mgl−1 to control Agrobacterium. Thus, 10mgl−1 kanamycin and 200mgl−1 cefotaxime were selected as optimal for genetic transformation. Agrobacterium-mediated genetic transformation was carried out with a GUS gene under the control of the chrysanthemum RbcS promoter. Two days of pre-culture and 5min of infection were found to result in the most efficient transformation rates. Ninety-nine percent of leaf explants infected with Agrobacterium showed GUS expression 3–5 days after transfection and 12% of the regenerated shoots had a stable GUS-positive phenotype. The other plants had a transient kanamycin-resistant phenotype and eventually lost the NPT-II and GUS gene expression.

Scientific Opinion on an application (EFSA-GMO-NL-2009-70) for the placing on the market of genetically modified drought tolerant maize MON 87460 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

Maize MON 87460 was developed through Agrobacterium-mediated transformation and expresses the cold shock protein B (CspB) from Bacillus subtilis and neomycin phosphotransferase II (NPTII) from Escherichia coli to reduce yield loss under water-limited conditions. Maize MON 87460 contains a single copy of the cspB and nptII expression cassettes. Bioinformatic analysis of the flanking sequences and the open reading frames spanning the junctions created by the transformation did not raise safety issues. Comparative analyses established that, besides the expression of the CspB and NPTII proteins, some differences were observed in the composition of forage and grain produced from maize MON 87460 compared with its conventional counterpart, when grown under well-watered conditions. Given the magnitude of these changes and the characteristics of these endpoints, the EFSA GMO Panel concluded that the observed differences do not raise safety concerns for humans and animals. Under stressful conditions, maize MON 87460 can show enhanced agronomic performance characteristics and some differences in chemical composition in comparison with its conventional counterpart. Given the intended trait, the observed differences were not unexpected, and did indicate no safety concerns. The safety assessment identified no concerns regarding the potential toxicity and allergenicity of the CspB and NPTII proteins, or of maize MON 87460. Maize MON 87460 is as nutritious as any other maize and can be used in the same way. In cases of spillage, there are no indications of increased likelihood of the establishment or survival of feral maize plants MON 87460. Risks associated with a theoretically possible horizontal gene transfer from maize MON 87460 to bacteria have been analysed in detail, including different scenarios of integration, and did not raise safety concerns for the intended uses of maize MON 87460. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 87460.

Establishment of a Genetic Transformation System for the Marine Pennate Diatom Fistulifera sp. Strain JPCC DA0580—A High Triglyceride Producer

A genetic transformation system for the marine pennate diatom, Fistulifera sp. JPCC DA0580, was established using microparticle bombardment methods. Strain JPCC DA0580 has been recently identified as the highest triglyceride (60% w/w) producer from a culture collection of 1,393 strains of marine microalgae, and it is expected to be a feasible source of biodiesel fuel. The transformation conditions for strain JPCC DA0580 were optimised using the green fluorescent protein gene (gfp) and the gene encoding neomycin phosphotransferase II (nptII). The most efficient rate of transformation was attained when tungsten particles (0.6μm in diameter) were used for microparticle bombardment. The effect of endogenous and exogenous promoters on the expression of nptII was examined. Endogenous promoters were more efficient for obtaining transformants compared with exogenous promoters. Southern hybridisation analysis suggested that nptII integrated into the nuclear genome. This genetic manipulation technique should allow us to understand the mechanisms of high triglyceride accumulation in this strain, thereby contributing to improving BDF production.

Genetic transformation of Pseudochoricystis ellipsoidea, an aliphatic hydrocarbon-producing green alga

Pseudochoricystis ellipsoidea is a recently isolated unicellular green alga, which is classified within the family Trebouxiophyceae. This alga has a unique ability to synthesize and accumulate intracellularly a significant amount of aliphatic hydrocarbons. To elucidate molecular mechanisms of the hydrocarbon production in this organism, the development of genetic methods including DNA transformation methods are important. Towards the goal, we constructed several plasmids in which neomycin phosphotransferase II-encoding G418-resistant gene (nptII) is flanked by a P. ellipsoidea-derived promoter and terminator. These plasmids were introduced into P. ellipsoidea cells through particle-gun bombardment, and transformants were screened among G418-resistant cells by PCR amplification of plasmid-borne genes. Southern blot analysis demonstrated that the exogenous DNA was integrated into the genome of the transformants. Furthermore, the expression of nptII was confirmed at the transcript and protein levels by RT-PCR and immunoblot analyses, respectively. These results clearly indicated that a genetic transformation system was successfully established for P. ellipsoidea.

Scientific Opinion on application EFSA-GMO-RX-MON1445 for renewal of the authorisation for continued marketing of cottonseed oil, food additives, feed materials and feed additives produced from cotton MON 1445 that were notified as existing products under

This scientific opinion evaluates the risk assessment for the authorisation for continued marketing of genetically modified herbicide tolerant cotton MON 1445 for food and feed produced from it. Cotton MON 1445 contains single copies of functional CP4 epsps and nptII expression cassettes and an aadA gene as non-functional element. Stability of the inserted DNA was confirmed over several generations. Bioinformatic analyses and levels of recombinant proteins did not reveal safety issues. No biologically relevant differences were identified in the compositional, phenotypic and agronomic characteristics of cotton MON 1445 in comparison to its conventional counterpart and its composition fell within the range of non-GM cotton varieties, except for CP4 EPSPS and NPTII proteins. No toxicity and allergenicity issues were identified regarding the newly expressed protens. Products from cotton MON 1445 do not contain viable plant parts. Insert structure in cotton MON 1445 may facilitate the stabilisation of the nptII gene in plasmids of environmental bacteria through double homologous recombination. However, considering the expected low frequency of gene transfer from cotton MON 1445 to bacteria compared to that between bacteria, and the very low exposure to DNA from cotton MON 1445, the EFSA GMO Panel concludes that the contribution of horizontal gene transfer to the environmental prevalence of nptII genes is negligible. Potential interactions of cotton MON 1445 with non-target organisms and the abiotic environment were not considered to be an issue because of low exposure levels. A post-market environmental monitoring plan is not required. The EFSA GMO Panel concludes that the information available for cotton MON 1445 addresses the questions raised by the Member States and that MON 1445-derived products are as safe as products derived from the conventional counterpart in the context of their intended uses.

Scientific Opinion on application EFSA‐GMO‐RX‐MON531 for renewal of the authorisation for continued marketing of existing cottonseed oil, food additives, feed materials and feed additives produced from MON 531 cotton that were notified under Articles 8(1)(a), 8(1)(b) and 20(1)(b) of Regulation (EC) No 1829/2003 from Monsanto

This scientific opinion evaluates the risk assessment for the authorisation for continued marketing of genetically modified insect resistant cotton MON531 for food and feed produced from it. MON531 was transformed via Agrobacterium. It contains single copies of functional Cry1Ac and NPTII expression cassettes; two fragments of the Cry1Ac cassette and aadA as non-functional elements. Stability of the inserted DNA was confirmed over several generations. Bioinformatic analyses and the levels of recombinant proteins did not reveal safety concerns. Analysis of compositional, phenotypic and agronomic characteristics indicated that MON531 is not different from its conventional counterpart and is compositionally within the range observed among conventional cotton varieties, except for Cry1Ac and NPTII. Safety assessment of Cry1Ac and NPTII proteins and cotton MON531 identified no concerns regarding potential toxicity and allergenicity. Products from MON531 do not contain viable plant parts. The aadA and oriV sequences in MON531 may facilitate the stabilisation of nptII through double homologous recombination in plasmid sequences in the environment. However, considering the expected low frequency of gene transfer from MON531 to bacteria compared to that between bacteria, and the very low exposure to MON531 DNA, the GMO Panel concludes that gene transfer from MON531 to bacteria does not indicate a risk to human or animal health or to the environment in the context of its intended uses. The exposure of potentially sensitive non-target organisms to Cry1Ac protein is likely to be low and of no biological relevance. A PMEM plan is not required. The EFSA GMO Panel considers that information available for cotton MON531 addresses the questions raised by the Member States and that cotton MON531, as described in this application, is as safe as its conventional counterpart and is unlikely to have adverse effects on human and animal health and the environment in the context of its intended uses.

Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration

An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.

Nucleosome positioning in the NPTII neomycin phosphotransferase gene on a yeast plasmid in the repressed and active states

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5′-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.

Expression of EGFP and NPTII Protein Is Not Associated with Organ Abnormalities in Deceased Transgenic Cloned Cattle

Both enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase type II enzyme (NPTII) are widely used in transgenic studies, but their side effects have not been extensively investigated. In this study, we evaluated the expression profiles of the two marker genes and the relationship between their expression and organ abnormalities. Eight transgenic cloned cattle were studied, four harboring both EGFP and NPTII, and four harboring only the NPTII gene. Four age-matched cloned cattle were used as controls. EGFP and NPTII expression were measured and detected by Q-PCR, Western blot, ELISA, and RIA in heart, liver, and lungs, and the values ranged from 0.3 to 5 microg/g. The expression profiles exhibited differential or mosaic pattern between the organs, the pathologic symptoms of which were identified, but were similar to those of age-matched cloned cattle. All data indicated that the expression of EGFP and NPTII is not associated with organ abnormalities in transgenic cloned cattle.

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens.

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.

Transfer and expression of nptII and bar genes in cucumber (Cucumis satavus L.)

The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l−1 phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l−1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l−1) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.

Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures.

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.

Increased Transgene Expression by Breeding and Selection in White Clover

To determine if standard breeding methodology is applicable to transgenes, phenotypic recurrent selection was used to select for increased transgene expression in white clover, Trifolium repens L. Plants were transformed with nptII and gusA, and selected on 100 mg L−1 of kanamycin. Independently transformed plants were intercrossed, and the progeny was germinated on 200, 300, or 400 mg L−1 of kanamycin. Those seedlings surviving on 400 mg−1 were in turn intercrossed, and the progeny was selected on 300, 400, or 500 mg L−1 of kanamycin. NPTII levels were measured in each selected population, and Southern blots were made from individuals in each population. The highest‐expressing individual in the T2 had levels of NPTII that were more than four times higher than those in the highest parent. With selection on increasing levels of kanamycin, average expression across each generation went from 0.033 ng μg−1 NPTII in the parents to 0.095 ng μg−1 in the selected T1 plants to 0.539 ng μg−1 in the selected T2 plants. Southern hybridization suggested that plants displaying a heightened level of nptII expression in the T1 and T2 fell into two categories. The first contained one particular transgenic event, implicating the importance of other genomic factors in modulating gene expression. Alternatively, the plants had an accumulation of various nptII loci, suggesting an association between multiple transgene copies and high expression levels. On the basis of these results, selection for transgene expression appears to be a viable option for plant breeding programs.